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Photosynthesis is a primary target of different abiotic stresses. Under sub-optimal conditions, the imbalance between a low-efficiency carbon fixation and excessive light absorbed can generate an overburdening of the photosynthetic apparatus. This can lead to the production of reactive oxygen species (ROS), which can damage cellular components. Plastid Terminal Oxidase (PTOX) has been proposed to play a role in some higher plants, acting as a safety valve for electrons, resulting in protection of the plastoquinone pool from over-reduction under environmental stress conditions. Due to PTOX ability to transport electrons from plastoquinol to molecular oxygen, generating water. The regulation of the enzyme is still unclear, however, it has been hypothesised to be sensitive to stromal pH variation and recent findings suggest that a translocation of the protein to the grana membranes could be involved. The protective role of PTOX as alternative electron sink in Hordeum vulgare plants was explored, in particular during cold acclimation and against water limitation and high salinity conditions. Additionally, the regulation of the activity of this enzyme was studied. Plastid terminal oxidase (PTOX) seems to account for the diversion of up to 25% of electrons to oxygen in control plants, acting as an important safety valve for electron transport potentially protecting barley leaves from an overreduction of the plastoquinol pool. In cold acclimated plants, PTOX does not appear to be important, however PTOX protein and transcript were still detected. In water restricted and salt treated plants, evidence of PTOX acting as a safety valve for electrons was seen. This plastoquinone oxidase seem to be diverting up to 31% of the total amount of electron transported by PSII at 21% O2 in water restricted plants and up to 45% in salt-treated leaves. This was supported by the presence of increased PTOX protein in both cases and also an increase in PTOX transcript either transiently, in water restricted plants, or in a sustained way in salt treated plants. Additionally, a dynamic and reversible response to O2 concentration was seen in the photosystem (PSII) electroon transport chain (ETR) and non-photochemical quenching (NPQ). Therefore it seems to be unlikely that PTOX is inhibited or in some way regulated by a previous oxygen concentration. It was not possible to confirm the translocation of PTOX to the grana in barley plants, however, direct or indirect variation in the pH has an effect on PTOX protein recovery in the salt-treated plants, with increasing detection of PTOX at more alkaline pH.

Most plants complete their life cycle in a single location and therefore are affected by the changing environment. As a result, plants have evolved physiological and developmental adaptations to overcome stress. The work presented in this thesis has examined the regulation of photosynthetic electron transport in barley, rice and Thellungiella salsuginea. Barley is considered as a crop which is comparatively tolerant to soil salinity. The focus of this study was to evaluate the physiological responses of photosynthesis in barley under salinity and to characterize traits responsible for the regulation of photosynthesis. At low salt concentrations, barley plants protect PSII centres from excitation pressure by down-regulating the electron transport chain and maintaining ΔpH, by cyclic electron transport associated with PSI, to support nonphotochemical quenching (NPQ). However, at the highest concentration of salt examined, this regulation starts to fail. The failure might result from a specific loss of PSI, resulting in reduced cyclic electron flow, or an increase in the leakiness of the thylakoid membranes, resulting in loss of ΔpH. The effects of salinity on the regulation of electron transport through Photosystem I and Photosystem II have been studied in two rice varieties from Sri Lanka. The regulation of photosynthesis in the salt-tolerant At-354 is more prominent than in the salt-sensitive Bg-352 when plants are exposed to salt. Exposure of Bg-352 to salt resulted in a substantial decrease in gas exchange, PSII photochemistry, leaf area and loss of chlorophylls. The decrease in the photosynthesis in AT-354 is caused by stomatal limitations, which restrict the CO2 entry into the plants, whereas the decrease of photosynthesis in Bg-352 is caused by non-stomatal limitations. Results suggest that At-354 protects PSII centres from excitation pressure by down-regulating the electron transport chain and maintaining ΔpH by cyclic electron transport associated with PSI to support NPQ. At high salt concentration, this regulation starts to fail in Bg-352.Tolerance to abiotic and biotic stress has evolved in many wild plant species, termed extremophiles. These plants contain essential genes which may used to improve crop production in changing environments. Thellungiella salsuginea is an extremophile, able to grow and reproduce in extreme environments. Stepien and Johnson (2009) identified a protein, known as the plastid terminal oxidase (PTOX) which acts as an alternative electron sink in T. salsuginea under salt stress. The current study showed that, in addition to salt, T. salsuginea showed increases in PTOX protein content and activity when exposed to drought, different growth irradiances and cold with high light. Semi-natural conditions also triggered the activity of PTOX. This study also showed that salt caused an up-regulation of PTOX gene transcripts in the leaves of salt treated T. salsuginea plants compared to control plants. Direct electron transport from PSII to PTOX and then to oxygen via the PQ pool accounted for up to 30% of total PSII electron flow in T. salsuginea (Stepien and Johnson, 2009). Efficient electron flow from PSII to PTOX would however, probably require co-location of these complexes in the same thylakoid fraction. To examine the location of PTOX in the thylakoid membrane, immunoblot analyses were performed, to test for changes in other protein complexes which may be associated with PTOX. In addition blue-native polyacrylamide gel electrophoresis and immunoblots were performed to isolate and detect the PTOX protein with any associated complexes. Although immunoblot analysis showed a prominent signal, mass spectrometry data did not allow identification of PTOX. This results suggests that further studies are needed to identify the precise localisation of the PTOX protein in the thylakoid membranes in T. salsuginea.

Chloroplast transformation technology is an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants, enabling them to be used as cellular factories. However more work is required to test this technology for the expression of membrane proteins, purification of recombinant proteins from the chloroplasts and high throughput cloning strategies. This work was undertaken with a view to expand the utility of chloroplast transformation technology. In the first part of the thesis, the potential of chloroplasts to express various membrane proteins was explored. The test proteins included a plastid terminal oxidase from Chlamydomonas reinhardtii (Cr-PTOX1) and a human G-protein coupled-receptor, adenosine receptor A (A2AR). Both proteins were successfully expressed in tobacco chloroplasts and in the case of Cr-PTOX1 in an active conformation. However, its transplastomic expression rendered the plants sensitive to high light. Interestingly protection was observed at the germination stage against salt stress in Cr-PTOX1 seedlings. The A2A receptor was also expressed; however it was not targeted to thylakoids. In the second part of the project, the expression in tobacco of two widely used affinity tags, glutathione S-transferase (GST) and His-tagged maltose-binding proteins (His6-MBP) was investigated as a mean of improving the purification of chloroplast expressed proteins. Their expression in transplastomic plant leaves reached approx. ≥7% and 37% of total soluble proteins, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His6-MBP by using a twin affinity purification procedure. Interestingly, the high level expression of GST led to cytoplasmic male sterility. Similarly, the feasibility of adapting the Gateway® cloning system for chloroplast transformation was also tested. The results suggested that Gateway® cloning does not give a high expression of recombinant proteins in tobacco chloroplasts. Overall this work opens up the opportunities for the expression of foreign membrane proteins in chloroplasts and expands the tools available for purifying recombinant proteins from the chloroplasts.